Proline rich dietary proteins such as caseins in bovine milk or glutens in cereals are known to resist proteolytic degradation in the human gastrointestinal tract. As a result proline rich peptides can build up and may lead to undesirable effects in specific groups of individuals. Some of these effects have been ascribed to the fact that the proline rich peptides act as opioids that bind to receptors in peripheral tissues and the central nervous system. For example, syndromes shown by autistic and schizophrenic patients have been linked with the consumption of proline rich dietary proteins. Other effects are the result of an intolerance for proline rich peptides. For example specific proline rich sequences are responsible for the observed toxicity of gluten in celiac disease. Celiac disease is a widely prevalent autoimmune disease of the small intestine which can only be treated by a life-long gluten free diet. Celiac disease is occasionally also accompanied by psychiatric and neurological symptoms illustrating the far-reaching consequences a disturbed metabolism of proline rich peptides may have.
Proteins in bovine milk are associated with growth and health and form an important ingredient in the human diet. Casein constitutes approximately 80% of the total protein in bovine milk and is an important source of amino acids, calcium and phosphate. Casein consists of roughly 50% of alpha-caseins, 35% of beta-caseins, 13% of kappa-caseins and 3% of gamma-caseins. In human milk the alpha-casein fraction is generally absent.
It is known that upon metabolisation of casein a number of new bioactive peptides are formed. From the alpha and beta-casein fractions opioid peptides called alpha-casomorphins and beta-casomorphins, respectively, have been identified and isolated. The pharmacological effects of especially the beta-casomorphins have been extensively studied. The beta-casomorphin with the sequence Tyr-Pro-Phe-Pro-Gly-Pro-Ile (SEQ ID NO:15) is the principal opioid peptide in bovine milk and is called BCM-7 (beta-casomorphin (1-7); Chang et al. (1985) Journal of Biological Chemistry, 260, 9706-9712). Apart from this BCM-7 fragment at amino acid positions 60-66 of the beta-casein molecule, smaller fragments of BCM-7 like Tyr-Pro-Phe-Pro (beta-casomorphin (1-4)) (SEQ ID NO:16) and Tyr-Pro-Phe-Pro-Gly (beta-casomorphin (1-5)) SEQ ID NO:17) at amino acid positions 60-63 and 60-64 respectively as well as all larger BCM-7 related peptides up to a chain length of 11 amino acids (at amino acid positions 60-70) display at least some degree of opioid activity. The N-terminal tripeptide of BCM-7, i.e. the sequence Tyr-Pro-Phe at position 60-62, has no opioid activity. A genetic beta-casein variant called A1 (having a histidine rather than the proline residue of A2 beta-casein at amino acid position 67) is claimed to lead to the formation of increased levels of the BCM-7 molecule.
The basic reason for the generation of the various beta-casomorphins is that their amino acid sequence is relatively rich in proline residues. Because peptide bonds involving proline residues resist proteolytic breakdown, the beta-casomorphin sequences tend to survive exposure to the gastrointestinal proteases in the stomach and the intestinal lumen. For the same reason one may assume that these beta-casomorphin sequences tend to survive incubations with other proteases, for example those proteases commonly used in the industrial production of protein hydrolysates. This assumption implies that the commonly available protein hydrolysates or products containing these protein hydrolysates all contain the BCM-7 or closely related peptides. As the BCM-7 peptide fragment and its related molecules have been linked with certain diseases, the presence of such molecules in protein hydrolysates, quite often used in the diet of vulnerable groups like infants, elderly and patients, is an undesirable situation. Results of opiate receptor binding assays of human and bovine beta-casomorphins indicate that the fragments with opioid activity bind with opiate receptors in the rat brain membrane. It has been shown that the beta-caseins are more selective towards mu-ligands with little affinity for delta- and kappa-receptor subtypes. According to these and other studies beta-casomorphins are claimed to have various gastrointestinal, analgesic, respiratory, cardiovascular, endocrine and immunomodulatory effects. A common structural feature of opioid peptides incorporating a proline residue is the Tyr-Pro-Phe/Trp motif (Okada et al, Vitamins and Hormones 2002, 65, 257-279).
Although in normal individuals the peptidases in the intestinal epithelial layer and in the blood can cope with the beta-casomorphins, this seems not to be always the case for patients suffering from schizophrenia, autism, ADHD or other mood disorders. For example, genetic alterations in plasma dipeptidyl peptidase IV (DPP IV) enzyme activity leading to an incomplete breakdown of proline rich peptides have been linked with the occurrence of these diseases. Moreover hyperpeptiduria, i.e. an increased concentration of casein or gluten derived peptides in the urine, is regularly found (Reichelt, W. H. et al; (1997) Dev. Brain Dysfunct; 10: 44-55). Recent scientific literature provides compelling evidence that an incomplete degradation of proline rich peptides may contribute to the development and the severity of such diseases. Apart from the caseine derived BCM-7 fragment, also gluten derived protease resistant peptides have been mentioned in this connection. Already in 1979 Panksepp (Trends in Neuroscience 1979; 2:174-177) proposed the opioid excess theory in which he suggested that a disturbed opioid metabolism is part of the pathogenesis in autism. Nowadays we understand that many proline rich peptides are highly resistant to cleavage by gastric and pancreatic peptidases such as pepsin, trypsin, chymotrypsin and the like and that only specific enzymes, as present in amongst others the brush border epithelial layer of the gastrointestinal tract, are capable of hydrolysing peptide bonds involving proline.
Gluten is the insoluble protein fraction of cereals like wheat, rye, oats, barley, maize and rice that remains after washing to remove starch and water-soluble components. Gluten can be subdivided into 4 major solubility fractions i.e. albumin, globulin, prolamin and glutelin. Among these especially the prolamin and the glutelin fractions of wheat, corn, barley and oats are characterized by relatively high contents of the amino acids proline and glutamine. Recent evidence has implicated the proline rich gluten sequences as a major factor in the development of celiac disease. Celiac disease, also known as celiac sprue, is an autoimmunedisease of the small intestine caused by the ingestion of gluten proteins. It commonly appears in early childhood with severe symptoms like chronic diarrhea and abdominal distension; later in life symptoms include fatigue, weight loss due to malabsorption and neurological symptoms. Among the proline rich fractions of the various cereals, alpha-gliadin from wheat, hordein from barley, secalin from rye and avenin from oats seem to be most toxic (Schuppan, D.; Gastroenterology 2000; 119:234-242). A life-long gluten free diet is the only effective treatment for celiac disease patients. Among celiac patients a high prevalence of various autoimmune disorders, especially type 1 diabetes, dermatitis herpetiformis, autoimmune thyroiditis, collagen diseases, autoimmune alopecia and autoimmune hepatitis has been observed. This indicates that by unknown mechanisms untreated celiac disease predisposes to autoimmunity to other organs (Schuppan, D. 2000 Gasteroenterology 119:234-242). Furthermore there are indications that a mild form of celiac disease is present in a group of people suffering from irritable bowel syndrome (IBS). IBS is a disorder that interferes with the normal functions of the large intestine and is characterized by crampy abdominal pain, constipation and diarrhea. IBS usually begins around the age of 20 and causes a great deal of discomfort and distress. The eating of wheat, barley, rye or milk products has been associated with a worsening of IBS symptoms.
Recently Shan et al (Science; vol 297, 27 September 2002: 2275-2279) identified a gliadin-derived, proline rich, 33 amino acids long peptide thought to be the source of a set of major celiac patient-specific T cell epitopes. Whereas an enzyme extract prepared from small intestine brush-border cells was unable to hydrolyse this 33-mer, suppletion with a bacterial prolyl oligopeptidase from Flavobacterium meningosepticum led to a rapid digestion with a concomitant strongly decreased stimulation of a relevant Tcell clone. In imitation of earlier work on the oral administration of papain (Messer, M. and Baume, P. E.; Lancet 1976; 2:1022), the article indicates the potential of the prolyl oligopeptidase as a dietary enzyme in detoxifying gluten by enzyme therapy.
Prolyl oligopeptidases (EC 3.4.21.26) have the unique possibility of preferentially cleaving peptides at the carboxyl side of proline residues. In the prolyl oligopeptidases isolated from mammalian sources as well as in the prolyl oligopeptidase isolated from Flavobacterium meningosepticum a unique peptidase domain has been identified that excludes large structured proteins from the enzyme's active site. In fact these enzymes are unable to degrade proteins containing more than about 30 amino acid residues so that these enzymes are now referred to as “prolyl oligopeptidases” (Fulop et al: Cell, Vol. 94, 161-170, Jul. 24, 1998). All known prolyl oligopeptidases are cytosolic enzymes that exhibit pH optima near neutrality and are characterized by the fact that they cannot efficiently degrade molecules containing more than approximately 30 amino acid residues. The fact that these enzymes exhibit pH optima that correspond with the pH values prevailing in the more distal part of the gastrointestinal tract, makes them ideally suitable as dietary supplements supporting the intestinal digestion process of dietary gluten.
Another enzyme that can have a benefit in the inactivation of toxic proline rich peptides, is the enzyme dipeptidyl peptidase IV (US2002/0041871A). Dipeptidylpeptidase IV, also called Xaa-Pro-dipeptidyl-aminopeptidase (EC 3.4.14.5) catalyzes the release of an N-terminal dipeptide, Xaa-Xbb from a peptide with the N-terminal sequence Xaa-Xbb-Xcc-, preferentially when Xbb is proline and provided Xcc is not proline. Dipeptidyl-peptidase IV has been isolated from a large number of mammalian sources, for example the intestinal brush border membranes form a rich source of the enzyme. Furthermore the enzyme has been isolated from microbial sources such as the food grade microorganisms Saccharomyces, Lactococcus and Aspergillus. Like the prolyl oligopeptidases, all known dipeptidyl-peptidases IV are enzymes with near neutral pH optima and thus suited for supporting the intestinal digestion process.
Because of the possible implications of the proline specific oligopeptidase and the dipeptidyl-peptidase IV in the treatment of celiac disease or schizophrenia, autism or other mood disorders, these data have resulted in a number of patent applications that deal with various aspects of this matter. For example U.S. Pat. No. 6,447,772 and WO 01/24816 describe compositions containing dipeptidyl peptidase IV, WO 03/068170 describes compositions containing proline specific oligopeptidases optionally combined with dipeptidyl-peptidase IV, WO 02/45523 describes low allergenic protein hydrolysates prepared with proline specific endoproteases and WO 03/028745 describes compositions comprising bacterial strains that can lower the concentration of intestinal toxic proline rich peptides. WO 96/36239 describes the advantages of products derived from cattle substantially free of the beta-casein A1 allele.